Summary

Diacylglycerol is an essential second messenger in mammalian cells. The
most prominent intracellular targets of diacylglycerol and of the functionally
analogous phorbol esters belong to the protein kinase C (PKC) family. However,
at least five alternative types of high-affinity diacylglycerol/phorbol-ester
receptor are known: chimaerins, protein kinase D, RasGRPs, Munc13s and DAG
kinase γ. Recent evidence indicates that these have functional roles in
diacylglycerol second messenger signalling in vivo and that several cellular
processes depend on these targets rather than protein kinase C isozymes. These
findings contradict the still prevalent view according to which all
diacylglycerol/phorbol-ester effects are caused by the activation of protein
kinase C isozymes. RasGRP1 (in Ras/Raf/MEK/ERK signalling) and Munc13-1 (in
neurotransmitter secretion) are examples of non-PKC
diacylglycerol/phorbol-ester receptors that mediate diacylglycerol and
phorbol-ester effects originally thought to be caused by PKC isozymes. In the
future, pharmacological studies on PKC must be complemented with alternative
experimental approaches to allow the separation of PKC-mediated effects from
those caused by alternative targets of the diacylglycerol second messenger
pathway. The examples of RasGRP1 and Munc13-1 show that detailed genetic
analyses of C1-domain-containing non-PKC
diacylglycerol/phorbol-ester receptors in mammals are ideally suited to
achieve this goal.

Introduction

Diacylglycerols (DAGs) are glycerol derivatives in which two hydroxyl
groups are substituted by fatty acids through ester bond formation. The
physiologically relevant isomer is 1,2-diacyl-sn-glycerol of which
mammalian cells contain many structurally distinct species that differ with
respect to the type and degree of saturation of their fatty acid moieties.
Ubiquitous DAGs are important intermediates in the synthesis and degradation
of triglycerides, glycerophospholipids and glyceroglycolipids. De novo
synthesis of DAG takes place in the endoplasmic reticulum.

Under equilibrium conditions, biological membranes contain very little
DAGs. Their production is stimulated upon activation of a multitude of
cellular signalling cascades, and DAGs produced by these mechanisms act as key
second messengers to modulate the function of at least six different types of
target protein, the most prominent of which belong to the protein kinase C
(PKC) family (see below) [for reviews of DAG signalling see
(
Wakelam, 1998;
Hodgkin et al., 1998;
Goni and Alonso, 1999)].

Key enzymes in most of the DAG-generating signalling processes are the
members of the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C
family (PI-PLCβ, PI-PLCγ, PI-PLCδ, PI-PLCϵ). Depending
on the PI-PLC subtype, different cellular signalling molecules induce
enzymatic activity via specific cell-surface receptors (see below).

This enzymatic activity results in the hydrolysis of phosphatidylinositol
4,5-bisphosphate, which contains mainly polyunsaturated fatty acids, to
inositol 1,4,5-trisphosphate and polyunsaturated DAGs. It appears that these
polyunsaturated DAGs resulting from PI-PLC activity rather than the more
saturated forms generated by alternative enzymatic pathways (see below) are
most relevant as intracellular messengers targeting PKCs (for reviews, see
Wakelam, 1998;
Hodgkin et al., 1998). Whether
non-PKC DAG targets show the same preference for polyunsaturated DAGs is not
known but is likely, given the similarity in pharmacological characteristics
of the respective binding sites (for reviews see
Kazanietz et al., 2000;
Kazanietz et al., 2002). Polyunsaturated DAG second messenger molecules are
inactivated by the activity of diacylglycerol kinases
(
Wakelam, 1998;
Hodgkin et al., 1998).
Activation of PI-PLCβ isozymes is initiated by ligand binding to
G-protein-coupled receptors. Relevant receptor systems include metabotropic
receptors for classic neurotransmitters, monoamine receptors and receptors for
numerous peptide signalling molecules. Apart from the Gq typeα
subunits coupled to these receptors, certain Gβγ subunits
can also activate PI-PLCβ. PI-PLCγ activation involves
phosphorylation by growth-factor-activated receptor protein tyrosine kinases
or by non-receptor protein tyrosine kinases such as Lck/Fyn or c-Src. In the
latter case, tyrosine kinase activation is mediated by G-protein-coupled
receptors and involves Gsα or Giα subunits.
PI-PLCγ isozymes can also be activated in a
protein-tyrosine-kinase-independent manner, involving phospholipid-derived
second messengers. Activation of PI-PLCδ isozymes is triggered by
binding of Ca2+ to the EF-hand and C2 domains of
PI-PLCδ, followed by the association of the PH domain with
phosphatidylinositol 4,5-bisphosphate. PLCϵ contains an RA domain that
binds to Ras. Activation of Ras (e.g. following growth factor signalling)
leads to translocation of PLC-ϵ to the plasma membrane and enzyme
activation. In addition, PLCϵ is activated by Gα12 (for
a review, see
Rhee, 2001).

Apart from the PI-PLC pathway, DAGs are produced from phosphatidylcholine,
which predominantly contains saturated and mono-unsaturated fatty acids, by
two subsequent reactions involving phosphatidylcholine-specific phospholipase
D (PC-PLD) and phosphatidic acid phosphohydrolase (for reviews, see
Wakelam, 1998;
Hodgkin et al., 1998). As is
the case for PI-PLCs, the two mammalian PC-PLDs (PC-PLD1 and PC-PLD2) are
activated by a plethora of cellular signalling cascades, often involving
cell-surface receptors for signalling molecules (for reviews, see
Exton, 1998;
Liscovitch et al., 2000;
Cockcroft, 2001). However, the
saturated/mono-unsaturated phosphatidic acid intermediates generated by PC-PLD
activity appear to be mainly responsible for the cellular signalling events
that are triggered by PC-PLD activation, whereas the
saturated/mono-unsaturated DAGs resulting from the subsequent phosphohydrolase
reaction may be irrelevant for signalling, at least as far as activation of
PKCs is concerned. Thus, the activity of specific phosphatidic acid
phosphohydrolases may lead to signal termination by inactivating
saturated/mono-unsaturated phosphatidic acid
(
Wakelam, 1998;
Hodgkin et al., 1998). In
general, PC-PLD activation by cell-surface receptors is indirect and often
mediated by G-protein-coupled receptors and G-protein activation. Depending on
the cellular process, PC-PLD stimulation involves intermediate activation of
Arf- and Rho-type small GTPases as well as of PKCs α and β. In
addition, lipid-derived signalling molecules such as phosphatidylinositol
4,5-bisphosphate or oleate, which are the products of regulated kinase or
lipase activities, stimulate PC-PLD
(
Exton, 1998;
Liscovitch et al., 2000;
Cockcroft, 2001).

Two additional cellular pathways of DAG production, both utilizing
phosphatidylcholine, involve phosphatidylcholine-specific PLC (PC-PLC) and
phosphatidylcholine-ceramide cholinephosphotransferase (for a review, see
Wakelam, 1998). In both
cases, mostly saturated/mono-unsaturated DAGs are produced, the signalling
role of which is questionable (see above). Moreover, direct evidence for an
involvement of DAGs generated by these pathways in mammalian cellular
signalling events is sparse, particularly where signalling cascades that are
triggered by the activation of cell surface receptors are concerned.

Considering the various possible sources of DAGs in mammalian cells, it is
evident that polyunsaturated DAGs resulting from PI-PLC activity are the most
relevant DAG second messengers. Irrespective of the PI-PLC isozyme involved,
induction of enzymatic activity causes the formation of DAG and inositol
1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate, in turn, leads to the
mobilisation of Ca2+ from intracellular stores; DAG is able to bind
to C1 domains of a large number of proteins with diverse function.
As mentioned above, the most prominent DAG targets belong to the PKC family of
serine/threonine kinases. Binding of DAG, often in synergy with
Ca2+, leads to membrane translocation and activation of PKC
isozymes (
Newton, 1995;
Newton, 1997;
Newton, 2001). After
activation, PKCs are thought to regulate a multitude of intracellular
processes, ranging from cell proliferation to neurotransmitter and hormone
secretion. Modulation of cellular processes by DAG and by the functionally
analogous phorbol esters (natural diterpene secondary metabolites of
Euphorbiaceae and Thymelaceae, see below) has often been
attributed exclusively to activation of PKCs. This is surprising because most
eukaryotic cells contain five alternative types of DAG targets [chimaerins,
protein kinase D1 (PKD1), RasGRPs, Munc13s and DAG kinase γ
(
Fig. 1) (for reviews see
Kazanietz, 2000;
Kazanietz, 2002;
Kazanietz et al., 2000)], and
the pharmacological tools that are frequently used to study PKC function are
not sufficiently specific to exclude the involvement of other DAG targets in
cellular processes that are thought to be mediated by modulatory effects of
DAG or phorbol esters on PKCs (
Betz et
al., 1998;
Kazanietz,
2000;
Kazanietz et al.,
2000;
Way et al.,
2000;
Rhee et al.,
2002). Indeed, a number of observations indicate that the effects
of DAG and phorbol esters are not mediated by PKCs but rather involve three
alternative DAG targets in at least three key cellular processes: (1) DAG- and
phorbol-ester-mediated subcellular translocation of PKD1 is essential for
protein transport from the trans-Golgi network to the cell surface
(
Matthews et al., 1999a;
Maeda et al., 2001;
Rey et al., 2001;
Baron and Malhotra, 2002;
Van Lint et al., 2002); (2)
activation of the Ras/Raf/MEK/ERK pathway in T lymphocytes is triggered by
G-protein-coupled receptors and tyrosine-kinase-coupled receptors and is
dependent on DAG-(or phorbol-ester-) induced activation of RasGRP rather than
PKCs (
Dower et al., 2000;
Jones et al., 2002); (3)
stimulatory effects of DAG and phorbol esters on neurotransmitter secretion
from nerve cells are mediated by DAG/phorbol-ester receptors of the
Unc-13/Munc13 family and not, as previously believed, by PKC isozymes
(
Betz et al., 1998;
Lackner et al., 1999;
Miller et al., 1999;
Nurrish et al., 1999;
Rhee et al., 2002).

As the relevance of non-PKC DAG receptors in the DAG second messenger
pathway is appreciated only by a rather small circle of experts in DAG
signalling but otherwise ignored, here we critique the widely accepted model
according to which all DAG/phorbol-ester effects are caused by the activation
of PKC isozymes, a view that is presented in most current textbooks and forms
the (at least implicit) conceptual basis of almost all past and current
pharmacological studies concerned with the determination of the role of PKC
isozymes in the control of cellular function. We briefly summarise established
and postulated functional roles of PKCs and discuss the characteristics of
pharmacological tools that are routinely used in most studies of PKC function
in vivo. Subsequently, we describe major caveats of pharmacological analyses
of PKC function and discuss alternative and more powerful experimental
approaches such as the use of dominant-interfering PKC variants, antisense
knockdown of PKC expression or PKC gene deletion in mice, which have yielded
important insights into PKC function but are not part of the methodological
repertoire in most studies of PKC function. We conclude with a discussion of
the functional importance of four non-PKC targets of the DAG second messenger
pathway, chimaerins, PKD1, RasGRPs and Munc13s — with a focus on the
latter.

PKCs as paradigmatic C1-domain-containing DAG
receptors

The protein sequence responsible for high-affinity binding of DAG and
phorbol esters was initially discovered in PKC isozymes and designated the
C1 domain. Depending on the PKC type, the C1 domain
consists of one (aPKCs) or two (cPKCs and nPKCs) zinc-finger-like repeats that
have a conserved pattern of cysteine and histidine residues and form a
coordination site for two Zn2+ ions
(H-X12-C-X2-C-X13/14-C-X2-C-X4-H-X2-C-X7-C).
Each individual zinc finger motif can form a single ligand-binding site for
DAG or phorbol ester. Only the single C1 domain motifs of aPKCs do
not bind DAG, and their function remains elusive
(
Mellor and Parker, 1998;
Newton, 2001). DAG or
phorbol-ester binding to the C1 domain does not significantly
change its conformation but rather creates a contiguous hydrophobic surface on
the side of the C1 domain that carries the ligand-binding pocket by
capping hydrophilic residues (
Zhang et
al., 1995) (for reviews, see
Hurley and Meyer, 2001;
Newton, 2001). The hydrophobic
surface of ligand-bound C1 domains then mediates membrane targeting
and concomitant activation of the corresponding enzymes. Apart from DAG
binding to C1 domains, full activation of cPKCs requires
Ca2+ and acidic phospholipids such as phosphatidylserine
(
Newton, 2001).
Ca2+-induced activation of cPKCs is mediated by the C2
domain. This region forms a compact β sandwich fold that binds
Ca2+ in a cup-shaped depression via five conserved aspartic acid
residues. Ca2+ binding is thought to trigger a conformational
change that opens a cleft to allow binding of an acidic phospholipid headgroup
of a membrane phospholipid, thus mediating membrane targeting and activation
of the corresponding PKC isozymes (
Mellor
and Parker, 1998;
Newton,
2001). nPKC C2 domains do not bind Ca2+ but
mediate essential protein-protein interactions (e.g. with substrates such as
GAP-43) (
Mellor and Parker,
1998;
Newton,
2001). In addition to C1 domains and C2
domains, the basic pseudosubstrate domains of cPKCs and nPKCs contribute to
membrane interactions during kinase activation
(
Newton, 1997).

In all cPKCs and nPKCs, kinase activation is closely coupled to ligand
binding by C1 and C2 domains and the resulting membrane
translocation, which in turn reverses autoinhibition caused by a
pseudosubstrate site. Essentially, the C1 and C2 domains
of cPKCs and the C1 domains of nPKCs can function as independent
membrane-targeting modules such that ligand binding by each individual domain
type leads to significant membrane association
(
Newton, 2001). Indeed,
phorbol ester or DAG binding to the C1 domains of nPKCs (and to a
lesser degree also of cPKCs) is sufficient for translocation and activation.
On the other hand, Ca2+-dependent phospholipid binding by the
C2 domains of cPKCs acts synergistically with DAG binding to the
respective C1 domain. Such synergistic activation of cPKC
C1 and C2 domains leads to the translocation and tight
membrane association of the enzyme, which then causes a conformational change
that reverts autoinhibition (
Newton,
2001;
Hurley and Meyer,
2001).

For proper cellular function of PKCs, their correct spatial distribution is
essential. Such spatially specific targeting of PKCs is unlikely to be brought
about by C1- and C2-domain-mediated membrane
interactions alone. Rather, specificity of PKC membrane targeting is thought
to be achieved by isozyme-specific binding proteins that are essential for the
formation of PKC-containing subcellular signalling complexes at the
appropriate subcellular location, thus spatially restricting PKC signalling
and allowing integration of PKC-mediated signalling with other intracellular
signalling pathways. PKC-binding proteins regulate targeting of PKC to
upstream activators (e.g. InaD, Syndecan-4), to substrates (e.g. STICKs,
RACKs) or to cytoskeletal and vesicular proteins (e.g. actin) (for reviews,
see
Csukai and Mochly-Rosen,
1999;
Jaken and Parker,
2000).

PKCs are thought to play essential roles in multiple cellular signal
transduction pathways of eukaryotic organisms. Genetic studies in yeast have
demonstrated that the dynamic regulation of the cell wall is the major
cellular site of PKC action in this simple organism. Deletion of PKC in
Saccharomyces cerevisiae leads to arrest of protein synthesis prior
to mitosis but after DNA synthesis. The underlying signalling pathway is
triggered by Hcs77p and involves the activation of PKC by Rho1. PKC
activation, in turn, initiates a phosphorylation cascade via a MAP kinase
module that leads to the activation of several transcription factors and
transactivation of, among others, heat shock and cell wall genes
(
Mellor and Parker, 1998).

Genetic studies on PKC function in mammals are more difficult to interpret
owing to the presence of multiple genes and possible functional redundancy. In
fact, all known PKC-deletion mutants show rather mild phenotypic changes.
Nevertheless, they have yielded important insights into the function of
individual PKC isozymes. (1) PKCγ, one of the most prominent PKC
isozymes in brain, has been shown to be important for brain functions involved
in learning and memory (Abelovic et al., 1993). Interestingly, the typical
stimulatory effects of phorbol esters on transmitter release are still
detectable in PKCγ-deficient nerve cells, indicating that alternative
DAG/phorbol-ester receptors are involved in this phenomenon
(
Goda et al., 1996). (2) Lack
of PKCβ leads to immunodeficiency [impaired humoral response and cellular
B cell response (
Leitges et al.,
1996)]. PKCβ appears to be critically involved in
B-cell-receptor-mediated survival signalling to NF-κB
(
Su et al., 2002).
Interestingly, B-cell-receptor-mediated signalling in PKCβ-deficient B
cells can still be bypassed by phorbol esters, indicating the involvement of
alternative DAG/phorbol-ester receptors in this pathway
(
Leitges et al., 1996). (3)
PKCϵ has been shown to be involved in the regulation of GABAA
receptor function (
Hodge et al.,
1999) and in the regulation of nociceptor function
(
Khasar et al., 1999). (4)
PKCθ appears to be involved in a unique signalling pathway linking T
cell antigen receptor signalling to NF-κB activation in mature T
lymphocytes (
Sun et al.,
2000). (5) PKCδ-deficient smooth muscle cells exhibit
increased apoptotic resistance (
Leitges et
al., 2001a). In addition, loss of PKCδ leads to increased
antigen-induced mast cell degranulation
(
Leitges et al., 2002) and to
the prevention of B cell tolerance owing to maturation and differentiation of
self-reactive B cells (
Mecklenbrauker et
al., 2002). (6) PKCζ is important for the regulation of
NF-κB transcriptional activity. As a consequence, lack of PKCζ
leads to impaired B cell receptor signalling, inhibition of cell proliferation
and survival and defects in the activation of ERK and the transcription of
NF-κB-dependent genes (
Leitges et
al., 2001b;
Martin et al.,
2002).

Numerous studies using alternative approaches indicate the involvement of
different PKCs in the modulation of ion channel conductance, transmitter
receptor function, smooth muscle contraction, cell migration, cell
proliferation and differentiation, apoptosis, lipogenesis, glycogenolysis, as
well as transmitter/hormone exocytosis and protein secretion [for examples
from the large number of reviews on PKC function in the literature, see
(
Kanashiro and Khalil, 1998;
Dempsey et al., 2000;
Barry and Kazanietz, 2001;
Ventura and Maioli, 2001)].
Apart from insights into PKC function that have been obtained in
pharmacological studies employing small molecule activators and inhibitors of
PKCs (which have particular advantages and disadvantages as discussed below),
many of the current models of PKC function originate from studies in which the
role of individual PKC isozymes was characterised using more informative
methodological approaches. These include the following: (1) overexpression of
wild-type and dominant interfering PKC mutants [e.g. PKCζ as a regulator
of RelA transcriptional activity (
Anrather
et al., 1999); PKCα and PKCδ as regulators of glucose
transport (
Tsuru et al.,
2002); PKCα and PKCθ as regulators of
calcineurin-induced transactivation (
Ishaq
et al., 2002); (for a review, see
Dempsey et al., 2000)]; (2)
interference with PKC expression using ribozymes [e.g. PKCα as a
regulator of glioma cell growth (
Sioud
and Sorensen, 1998)]; (3) interference with expression using
antisense oligonucleotides [for reviews of the literature with an emphasis on
therapeutically relevant approaches see
(
Tamm et al., 2001;
Goekjian and Jirousek, 2001;
Swannie and Kaye, 2002)]; and
(4) interference with PKC function using peptides that induce or block PKC
interactions with targeting proteins (for reviews, see
Csukai and Mochly-Rosen, 1999;
Jaken and Parker, 2000).

All the above approaches are conceptually and experimentally more stringent
than the classic pharmacological studies (see below), although they have their
individual caveats. Overexpression often results in levels of wild-type or
dominant-negative PKC variants that exceed endogenous levels by an order of
magnitude or more. As a result, overexpressed wild-type PKC isozymes may
participate in signalling processes that they are usually not involved in, and
mutant variants (e.g. kinase-deficient mutants) may interfere in a
dominant-negative manner with signalling to other targets of signalling
pathways (e.g. the DAG second messenger pathway). These problems can be
accounted for by complementing data obtained in overexpression studies with
data obtained using deletion mutations of the corresponding PKC isoform under
investigation. Ribozymes and particularly antisense oligonucleotides often
yield only partial knockdown of expression levels. Peptides, by contrast, are
often used at rather high concentrations such that non-specific effects must
be excluded. Irrespective of the distinct advantages of these experimental
approaches, the majority of studies of PKC function in different cell
biological processes are not characterised by a comparable conceptual and
experimental stringency. In most of these cases, which are typically concerned
with the problem of whether PKCs in general are involved in a given cellular
process, commercially available pharmacological tools are used to activate or
inhibit PKCs. The main caveat with these pharmacological studies is that
neither the almost exclusively used phorbol-ester-derived PKC activators nor
many of the commonly used PKC inhibitors are specific for PKCs (see
below).

Pharmacological tools to interfere with PKC function

DAG is one of the most important second messengers involved in PKC
activation, and certain DAG-related cyclic lactones are potent PKC activators.
However, the most commonly used pharmacological tools for PKC activation
belong to the phorbol-ester family of tumour promoters. Phorbol esters are
secondary metabolites of Euphorbiaceae and Thymeleaceae and
mimic the action of DAG at C1 domains. In common with DAG, phorbol
esters bind to the C1 domain of PKCs and induce membrane
translocation and activation of the enzyme (for reviews, see
Hurley and Meyer, 2001;
Newton, 2001;
Barry and Kazanietz, 2001). In
comparison with phorbol esters, efficacy and specificity of alternative PKC
activators such as thymeleatoxin, sapintoxins A and D, 12-deoxyphorbol esters,
mezerein, indolactam V, resiniferatoxin, tinyatoxin, thapsigargin or
bistratene A are much lower (
Way et al.,
2000). Unfortunately, none of the commonly used phorbol esters is
specific for PKCs. In fact, several alternative C1-domaincontaining
proteins, including the chimaerin (
Ahmed et
al., 1990;
Ahmed et al.,
1993;
Areces et al.,
1994;
Caloca et al.,
1997;
Caloca et al.,
2001), Munc13 (
Betz et al.,
1998) and RasGRP (
Ebinu et
al., 1998;
Lorenzo et al.,
2000) protein families, as well as PKD1
(
Valverde et al., 1994) and
DAG kinase γ (
Shindo et al.,
2001), bind phorbol esters with PKC-like affinity (see below) (for
reviews see
Ron and Kazanietz,
1999;
Kazanietz,
2000;
Kazanietz,
2002;
Kazanietz et al.,
2000;
Barry and Kazanietz,
2001). Thus, studies of PKC function that rely on the use of
phorbol esters as an investigative tool have to be interpreted with caution.
This is particularly pertinent for cellular processes that are also regulated
by alternative DAG/phorbol-ester receptors (e.g. the regulation of
intracellular vesicle transport by PKD1, the regulation of transcription by
RasGRPs or the regulation of neurotransmitter release by Munc13 isoforms).

As is the case for C1-domain-directed PKC activators,
C1-domain-directed PKC inhibitors are non-specific pharmacological
tools that bind with comparable affinity to other C1 domain
proteins. Such non-specific inhibitors include one of the PKC inhibitors used
most widely in the past, calphostin C
(
Betz et al., 1998; for a
review, see
Barry and Kazanietz,
2001). For the functional separation of PKC-specific effects from
those mediated by alternative DAG/phorbol-ester receptors, some of the most
useful pharmacological tools are ATP-binding site inhibitors. Although many of
these (e.g. the indolocarbazole staurosporin, some balanol analogs,
phenylaminopyrimidines, and rottlerin) inhibit protein kinases
non-specifically, certain indolocarbazoles (e.g. Midostaurin/CGP41251,
Gö6976, Gö7612, Gö7874, UCN-01) and bisindolylmaleimides
(Gö6850, Gö6983, LY-333531, LY-379196, LY-317615) are rather PKC
specific, some even show a preference for certain isozymes
(
Kanashiro and Khalil, 1998;
Barry and Kazanietz, 2001;
Way et al., 2000;
Goekjian and Jirousek, 2001;
Swannie and Kaye, 2002).

The main problem with some of the most specific bisindolylmaleimide-derived
PKC inhibitors is their partial toxicity in certain situations. Gö6859,
for example, causes a dramatic nonspecific rundown of synaptic transmission in
primary hippocampal nerve cells without affecting phorbolester effects in this
system (
Fig. 2)
(
Rhee et al., 2002). Given
that most PKC inhibitors are usually applied according to a preincubation
paradigm (i.e. for minutes) and at rather high concentrations, even mild
nonspecific or toxic effects of such drugs can have profound consequences.
Nevertheless, the indolecarbazol CGP41251/midostaurin and the
bisindolylmaleimide LY-333531 have advanced to late-stage clinical
developmental for the treatment of cancer and other indications, demonstrating
that these drugs act quite specifically under carefully controlled conditions
(for reviews, see
Goekjian and Jirousek,
2001;
Swannie and Kaye,
2002).

Nonspecific effects of a bisindolylmaleimide-derived PKC inhibitor in
hippocampal neurons. (A) Time course of phorbol-ester effects on evoked EPSCs
in wildtype neurons (n=6). Application of PDBU (1 μM) is indicated
by the white box. (B) Preincubation with 3 μM bisindoylmaleimide I
(Gö6857, grey box) led to a partially irreversible rundown of evoked EPSC
amplitudes but did not block the potentiation induced by application of 1μ
M PDBU (white box). (C) Average evoked EPSC amplitudes in untreated
neurons before and after bisindoylmaleimide I (3 μM) pretreatment
(n=6). (D) Average phorbol-ester-dependent potentiation of evoked
EPSC amplitudes (30 seconds following onset of application of 1 μM PDBU,
n=6) in untreated and bisindoylmaleimide I (3 μM) pretreated
neurons from experiments shown in (B). Error bars indicate s.e.m.

Apart from small molecule inhibitors of PKC, a recently emerged alternative
pharmacological approach to perturb PKC activity involves peptides that
interfere with the membrane translocation and targeting of PKCs by blocking or
inducing their interaction with anchoring proteins such as RACKs. Currently
PKC-isozyme-specific inhibitor and activator peptides for all cPKCs and nPKCs
are available. They have proven to be useful tools in dissecting signalling
processes mediated by individual PKC isozymes [e.g. in the context of PKC
effects in cardiac myocytes on contraction, ischemic cell death, MAP kinase
activation and ion channel activity (for reviews, see
Csukai and Mochly-Rosen, 1999;
Schechtman and Mochly-Rosen,
2002)]. Unfortunately, the use of PKC-isozyme-specific interfering
peptides has been restricted to a rather small number of studies. In the
majority of pharmacological studies on PKC function, the potential of the
peptide interference method has been ignored, and experimental approaches have
been limited to the default use of classic but often problematic
pharmacological tools (i.e. phorbol esters, indolocarbazoles, and
bisindolylmaleimides) described above.

An additional promising approach for interfering with PKC function involves
the use of antisense oligonucleotides to knockdown PKC expression. In
particular, downregulation of PKCα expression using antisense
oligonucleotides appears to have significant therapeutic potential
(
Dean and McKay, 1994) (for
reviews, see
Tamm et al.,
2001;
Goekjian and Jirousek,
2001;
Swannie and Kaye,
2002). Despite its high potential and usefulness for systematic
analyses of PKC function, the antisense approach is very rarely used in basic
research and largely ignored in the majority of pharmacological studies of PKC
function.

In summary, the most frequently used pharmacological tools for PKC
activation and inhibition (i.e. phorbol esters, indolocarbazoles and
bisindolylmaleimides) are not sufficiently specific to define PKC-mediated
physiological effects unequivocally in any experimental paradigm —
particularly the separation of PKC-mediated effects from those caused by other
C1 domain proteins or by other kinases remains difficult. In view
of the fact that most pharmacological studies of PKC function involve
phorbol-ester-mediated perturbations of certain cellular parameters, followed
by the addition of rather nonspecific PKC inhibitors, the involvement of
alternative DAG/phorbol-ester receptors in the observed effects must be
considered wherever phorbol esters are used as the main investigative tools.
PKC-isozyme-specific inhibitor and activator peptides and certain antisense
oligonucleoides are the most promising pharmacological tools to circumvent the
problems involved in the exclusive use of phorbol esters, indolocarbazoles and
bisindolylmaleimides to activate or inhibit PKCs. In addition, systematic
genetic studies represent an essential experimental alternative. The fact that
deletion mutations of PKCγ (Abelovic et al., 1993), PKCβ
(
Leitges et al., 1996),
PKCϵ (
Hodge et al., 1999;
Khasar et al., 1999),
PKCθ (
Sun et al.,
2000), PKCδ (
Leitges et
al., 2001a) or PKCξ
(
Leitges et al., 2001b) in
mice have rather mild phenotypic consequences indicates that there is
functional redundancy among the various PKC isozymes. To account for this
problem and ultimately to determine the role of individual PKCs, multiple
deletion mutations (e.g. of related PKC isozymes) may also be needed. Such
genetic approaches in intact animals could then be ideally complemented with
protein overexpression approaches.

Interestingly, all of the proven non-PKC DAG/phorbol-ester receptors
identified here function in intracellular signalling pathways that — at
least according to pharmacological evidence — are thought to be also
regulated by PKCs. Such functional overlap between PKC and non-PKC
DAG/phorbol-ester receptors is particularly evident in the case of the
chimaerins, which are thought to play a critical role in the regulation of the
actin cytoskeleton, cell cycle progression and malignant transformation (for
reviews, see
Ron and Kazanietz,
1999;
Kazanietz,
2000;
Kazanietz,
2002; Kazanietz et al., 2002). It is similarly evident in the case
of RasGRPs, which function in the control of cell proliferation,
differentiation and transformation by regulating the Ras/Raf/MEK/ERK pathway
(
Dower et al., 2000;
Jones et al., 2002), and in
the case of Munc13s, which are essential regulators of secretory vesicle
priming and transmitter/hormone release
(
Betz et al., 1998;
Rhee et al., 2002; for
reviews, see
Brose et al.,
2000;
Lloyd and Bellen,
2001). Given the limited specificity of the commonly used
PKC-directed pharmacological tools (particularly of the universally used
phorbol esters), the significant number of putative non-PKC DAG/phorbol-ester
receptors and the involvement of proven non-PKC DAG/phorbol-ester receptors in
cellular processes that have often been associated with PKC function in the
past, it is very likely that several of the identified phorbol-ester and DAG
effects in mammalian cells are in fact mediated by non-PKC DAG/phorbol-ester
receptors. This view is supported by a number of studies in which
pharmacological effects of phorbol esters and other PKC-directed drugs could
not be correlated with PKC function (e.g.
Scholfield and Smith, 1989;
Fabbri et al., 1994;
Simon et al., 1996;
Redman et al., 1997;
Stevens and Sullivan, 1998;
Hori et al., 1999;
Honda et al., 2000;
Iwasaki et al., 2000;
Waters and Smith, 2000). More
recently, several studies provided direct evidence for the functional
importance of the regulation of four non-PKC DAG/phorbol-ester receptors by
DAG and phorbol esters in distinct cellular processes.

Functional relevance of non-PKC DAG/phorbol-ester receptors as
targets of the DAG second messenger pathway

Chimaerins

Chimaerins were the first high-affinity non-PKC DAG/phorbol-ester receptors
to be discovered (
Hall et al.,
1990;
Ahmed et al.,
1990). They constitute a family of two isoforms (α andβ
) that are expressed from different genes, and each occurs as two splice
variants (1 and 2). The type 1 chimaerins contain a C1 domain,
followed by a Rac-GTPase-activating domain, whereas the type 2 chimaerins have
an additional N-terminal SH2 domain (
Fig.
1) (for reviews, see
Kazanietz, 2000;
Kazanietz, 2002;
Kazanietz et al., 2000).

In common with cPKCs and nPKCs, chimaerins translocate to phospholipid
membranes in response to phorbol-ester binding
(
Caloca et al., 1997).
Chimaerins are implicated in diverse cellular processes, such as cell adhesion
(
Herrera and Shivers, 1994),
cytoskeletal dynamics (
Herrera and
Shivers, 1994), lamellipodium/filopodium formation
(
Kozma et al., 1996),
phagocytosis (
Cox et al.,
1997), neuritogenesis and nerve cell development
(
Leung et al., 1994;
Hall et al., 2001). The
Rac-GTPase-activating function of chimaerins
(
Diekmann et al., 1991) is
likely to be involved in these processes but direct evidence for a function of
chimaerins in signalling to Rac in vivo is still lacking. Current information
on the function of chimaerins is mostly derived from overexpression studies,
and additional work using complementary methods is needed to verify an in vivo
role of chimaerins in the processes mentioned above.

The C1 domains of chimaerins are high-affinity DAG/phorbol-ester
binding sites (
Ahmed et al.,
1990;
Ahmed et al.,
1993;
Areces et al.,
1994;
Caloca et al.,
1997;
Caloca et al.,
2001), and chimaerins act as functional phorbol-ester receptors
when overexpressed in cells. β2-chimaerin, for example, translocates from
a cytosolic compartment to the plasma and Golgi membranes after phorbol-ester
treatment (
Caloca et al.,
2001;
Wang and Kazanietz,
2002). This translocation is dependent on an intact C1
domain and thought to be supported in vivo by an additional interaction with a
cis-Golgi transmembrane protein, Tmp21-I
(
Wang and Kazanietz, 2002).
However, phorbol esters do not (or do only very weakly) affect the
GTPase-activating function of chimaerins
(
Ahmed et al., 1993;
Kazanietz, 2002), which
indicates that the function of DAG binding is primarily to translocate
chimaerins to membranes, thus spatially restricting their
Rac-GTPase-activating effects. How this membrane translocation of chimaerins
relates to Rac signalling, particularly in the case of Golgi membranes, is
unknown (Kazanietz et al., 2002).

PKD1

The PKD family consists of PKD1 (also called PKCμ), PKD2 and PKD3 (also
called PKCν) (for a review, see
Van
Lint et al., 2002). These enzymes form a subfamily of the AGC
superfamily of serine/threonine kinases that is structurally related to but
distinct from other AGC superfamily members such as PKCs
(
Valverde et al., 1994;
Nishikawa et al., 1997;
Hayashi et al., 1999;
Sturany et al., 2001). PKD1,
the most prominent family member, contains an N-terminal apolar domain, two
C1 domains, a negatively charged central domain, a
pleckstrin-homology domain and a serine/threonine kinase domain
(
Fig. 1). PKD1 is activated by
multiple signalling mechanisms. A major PKD1 activation mechanism involves
protein phosphorylation by PKCϵ and/or PKCη, which is likely to be
triggered by G-protein-coupled receptors followed by activation of
PI-PLCβ and concomitant DAG production
(
Iglesias et al., 1998;
Matthews et al., 1999b;
Vertommen et al., 2000;
Waldron et al., 2001). This
functional interaction between PKD1 and PKCs, which is a striking example of a
mechanistic coupling between two types of DAG/phorbol-ester receptors, can be
triggered in vivo by neuropeptides (via a pathway involving G-protein-coupled
receptors and PI-PLCβ), growth factors (via activation of PI-PLCγ)
or even oxidative stress (via Src and PI-PLCγ) (for a review, see
Van Lint et al., 2002). In
addition, PKD1 is regulated by 14-3-3 proteins
(
Hausser et al., 1999),
Gβγ subunits (
Jamora et al.,
1999) and by caspase-mediated cleavage
(
Endo et al., 2000).

Although the corresponding evidence is in some cases still fragmentary and
often relies only on protein overexpression studies, PKD1 is thought to be
involved in the regulation of several cellular processes, including cell
proliferation (
Rennecke et al.,
1999), cancer cell invasion of tissues
(
Bowden et al., 1999) and
apoptosis (
Johannes et al.,
1998). The best characterised function of PKD1 is its regulatory
role in the Golgi apparatus, where it is required for transport vesicle
formation and transport of proteins from the Golgi apparatus to the plasma
membrane (
Jamora et al., 1999;
Liljedahl et al., 2001).
According to a current model, PKD1 is recruited to the Golgi apparatus by
binding of its first C1 domain to DAG in the Golgi membrane.
Together with effector proteins, PKD1 then forms a vesicle budding complex
that causes membrane deformation, formation of short tubules and finally
vesicle fission (for a review, see
Van
Lint et al., 2002). Binding of DAG to the first C1
domain of PKD1 is mainly involved in targeting and localisation of the kinase
(
Maeda et al., 2001;
Baron and Malhotra, 2002). In
addition, the C1 domains of PKD1 may be involved in the regulation
of its kinase activity (
Hausser et al.,
2002).

RasGRP1 and RasGRP3 have now been shown to be high-affinity
DAG/phorbol-ester receptors (
Lorenzo et
al., 2000;
Lorenzo et al.,
2001), and RasGRP1 translocates to membrane compartments in
response to phorbol-ester treatment (
Ebinu
et al., 1998;
Tognon et al.,
1998). In intact cells, RasGRP1 couples muscarinic acetylcholine
receptors (
Guo et al., 2001)
and T cell receptors (
Dower et al.,
2000;
Jones et al.,
2002) to the Ras/Raf/MEK/ERK pathway independently of PKCs.
Indeed, in Ras signalling assays and cell proliferation assays, mutant
thymocytes that lack RasGRP1 are insensitive to phorbol esters and T cell
receptor activation (
Dower et al.,
2000). These genetic data, together with evidence from
complementary studies described above demonstrate beautifully that DAG-induced
induction of the Ras/Raf/MEK/ERK pathway — at least in thymocytes—
is entirely dependent on RasGRP1 and unlikely to involve PKCs. This
discovery is particularly striking because numerous studies in different cell
types have related the activation of the Ras/Raf/MEK/ERK pathway to PKC
activity — in almost all cases relying on the conventionally used
pharmacological tools for PKC activation and inhibition, that is, phorbol
esters, indolocarbazoles, and bisindolylmaleimides (for reviews, see
Goekjian and Jirousek, 2001;
Ventura and Maioli, 2001).
The data obtained in RasGRP1 deleted mutant thymocytes provide the first
direct and convincing evidence for a cellular DAG signalling pathway that is
mediated by a non-PKC DAG/phorbol-ester receptor rather than by PKCs, as had
been thought previously. It is likely that thymocytes are not the only cell
type in which allegedly PKC-mediated effects on the Ras/Raf/MEK/ERK pathway
are in fact caused by RasGRPs.

All Munc13 isoforms bind phorbol esters and DAG with high affinity and—
in common with PKCs — translocate to the plasma membrane in
response to phorbol-ester binding (
Betz et
al., 1998;
Ashery et al.,
2000). As is the case for PKC C1 domains
(
Hommel et al., 1994;
Quest et al., 1994), mutation
of the first histidine residue in the Munc13-1 C1 motif to lysine
(H567K) abolishes DAG and phorbol-ester binding as well as
phorbol-ester-dependent membrane translocation
(
Betz et al., 1998). These
findings led to the hypothesis that Munc13 proteins are functional presynaptic
phorbol-ester receptors and targets of the DAG second messenger pathway that
act in parallel with PKCs to regulate transmitter release
(
Betz et al., 1998). This
hypothesis conflicted with numerous pharmacological studies that had
identified PKCs as the main mediators of phorbol-ester effects on transmitter
release from hippocampal neurons and that had established the concept that
PKCs are the only physiological DAG-dependent mediators of enhanced
neurotransmitter output that have a role in transient and long-term
potentiation of synaptic strength
(
Stevens and Sullivan, 1998)
(for a review, see
Majewski and Iannazzo,
1998).

The functional relevance of binding of DAG/phorbol esters to Munc13-1 in
vivo was determined in knockin mutant mice that express the
DAG/phorbol-ester-binding-deficient Munc13-1H567K mutant
instead of the wild-type Munc13-1 from the endogenous Munc13-1 locus
(
Rhee et al., 2002).
Homozygous Munc13-1H567K mutant mice die immediately after birth,
demonstrating that an intact Munc13-1 C1 domain is essential for
survival. Hippocampal nerve cells from homozygous
Munc13-1H567K mutants are almost completely insensitive to
phorbol esters, whereas wild-type cells show robust increases in transmitter
release in response to phorbol-ester treatment
(
Rhee et al., 2002). The
residual phorbol-ester sensitivity in homozygous
Munc13-1H567K cells is due to the presence of small
amounts of Munc13-2, as demonstrated by the complete lack of phorbol-ester
responses in cells that express Munc13-1H567K in a
Munc13-2 deletion mutant background
(
Fig. 3). Because expression
and function of PKCs is unaffected in Munc13-1H567K
mutants and Munc13-1 is not a substrate of phorbol-ester-activated PKCs
(
Rhee et al., 2002), these
genetic data indicate that the phorbol-ester-induced augmentation of
neurotransmitter release from hippocampal nerve cells is mediated exclusively
by Munc13 proteins and not by PKCs. PKCγ, one of the prominent PKCs in
synapses, has been shown not to be involved in mediating phorbol-ester effects
on transmitter secretion (
Goda et al.,
1996). Thus, Munc13s rather than PKCs are the only functionally
relevant, phorbol-ester-and DAG-sensitive presynaptic regulators of
transmitter release.

In view of the Munc13-1H567K mutant phenotype in
hippocampal neurons, it is possible that other documented effects of phorbol
esters on regulated secretory processes are also mediated by Munc13s rather
than by PKCs. In this context, future genetic studies will have to determine
whether published phorbol-ester effects on the release of catecholamines
(chromaffin cells), insulin (β cells), growth hormone (pituitary),
acetylcholine (neuromuscular junction) or dopamine (striatum) are mediated by
PKCs or Munc13s (
Kanashiro and Khalil,
1998).

The fact that homozygous Munc13-1H567K mutant mice die
immediately after birth demonstrates that Munc13-1 — in contrast to
individual PKC isoforms — is an essential functional target of the DAG
second messenger pathway in the brain. Detailed physiological analyses showed
that the replacement of wild-type Munc13-1 with a DAG-binding-deficient
Munc13-1H567K mutant leads to striking functional changes
in hippocampal nerve cells. Munc13-1H567K mutant cells
exhibit a reduction in the number of fusion-competent vesicles, a stronger
depression of synaptic transmitter release during high-frequency action
potential trains, and a reduction in the activity-dependent refilling of the
fusion competent vesicle pool (
Rhee et
al., 2002). These data indicate that DAG-dependent activation of
Munc13-1 allows nerve cells to adjust their vesicle priming machinery to
increases in activity levels. High-frequency stimulation and concomitant
Ca2+ influx or activation of presynaptic receptors appears to
activate PI-PLC isozymes (e.g. PI-PLCδ and PI-PLCβ) and thus lead
to transient increases in synaptic levels of DAG, which in turn binds to the
C1 domain of Munc13-1 and boosts its priming activity
(
Rhee et al., 2002;
Rosenmund et al., 2002). The
fact that Munc13-1H567K mutant mice die immediately after
birth indicates that the C1-domain-dependent stimulation of
Munc13-1 activity and the resulting adaptation to high-activity levels is
important for neurons involved in essential body functions (e.g. rhythmically
active nerve cells in the respiratory system).

A molecular model of how Munc13-1 activation by DAG regulates synaptic
efficacy during periods of high synaptic activity can be inferred from the
mechanism of DAG-dependent membrane recruitment of PKCs
(
Fig. 4). Munc13-1 is present
in a soluble pool and a pool that is tightly associated with the cytoskeletal
matrix of the presynaptic active zone by a proteinaceous linker
(
Betz et al., 2001), and
soluble Munc13-1 can translocate to the plasma membrane in a phorbol-ester
dependent (and presumably also DAG-dependent) manner
(
Betz et al., 1998;
Ashery et al., 2000). The
insoluble, active zone resident Munc13-1 is functionally integrated into the
release machinery of the active zone, has access to all necessary regulatory
proteins (
Betz et al., 2001)
and may define the basal pool of fusion competent vesicles that are
characterised by slow pool-refilling rates and high vesicular release
probability Pvr (
Rhee et al.,
2002). A second pool of fusion-competent vesicles that is
dependent on the Munc13-1 C1 domain and characterized by fast
refilling rates but low Pvr
(
Rhee et al., 2002) may be
generated by Munc13-1 molecules that have been recruited from the cytosol to
the presynaptic plasma membrane in a DAG-dependent manner. These recruited,
non-active-zone-resident Munc13-1 molecules could represent `ectopic' priming
sites that are partially functional but lack active-zone-specific regulatory
components, hence their fast refilling rate and low Pvr. In the
absence of stimulation, the number of `ectopic' priming sites would depend on
the resting DAG level in the presynaptic active zone plasma membrane.
Tonically present `ectopic' priming sites would be largely eliminated in
Munc13-1H567K neurons, leading to the observed reduction
in the size of the readily releasable vesicle pool
(
Rhee et al., 2002). The
number of `ectopic' sites would be increased by activity-dependent increases
in membrane DAG levels or by phorbol esters, which indeed cause increases in
the size of the readily releasable vesicle pool
(
Stevens and Sullivan,
1998).

Model of Munc13-1 activation by DAG. See text for details. Note that only
one mechanism of DAG synthesis (i.e. PI-PLCβ) is depicted here. Other
PI-PLC activities are likely to also activate Munc13s. Indeed, PI-PLCδ
may be responsible for activation of Munc13-1 during high-frequency
stimulation and intrasynaptic accumulation of Ca2+.

Conclusions

DAG is one of the most important second messengers in mammalian cells. The
functionally most relevant polyunsaturated DAG species are generated from
phosphatidylinositol 4,5-bisphosphate by PI-PLCs after activation of different
types of cell-surface receptors. The most prominent intracellular targets of
this DAG belong to the PKC family of serine/threonine kinases. However, owing
to their limited specificity, the pharmacological tools that are commonly used
to study PKC-mediated cellular processes (i.e. phorbol esters,
indolocarbazoles and bisindolylmaleimides) are not adequate to determine the
functional significance of PKC isozymes in defined cellular signalling
processes. As a consequence, the involvement of PKCs in a number of allegedly
PKC-mediated processes in mammalian cells remains to be proven by alternative
experimental approaches. These include the systematic use of
activating/inhibiting peptides, interference with PKC expression by antisense
oligonucleotides or RNAi, overexpression of wild-type and dominant-interfering
PKC forms and, most importantly, further genetic studies in mice. The
importance of such studies is evident from work showing that mammals express
at least five types of non-PKC high-affinity DAG/phorbol-ester receptor—
chimaerins, PKD1, RasGRPs, Munc13s and DAG kinase γ —
whose functions partially overlap with those of PKC isozymes. RasGRP1 (in the
context of DAG-dependent Ras/Raf/MEK/ERK signalling) and Munc13-1 (in the
context of DAG-dependent regulation of neurotransmitter release) mediate
DAG/phorbol-ester effects that had previously been attributed to PKCs. This
disproves the notion that all DAG/phorbol-ester effects are mediated by PKC
isozymes, a view that is widely accepted in the life sciences and represents
the implicit conceptual basis of most pharmacological studies on PKCs. Past
and future pharmacological studies on protein kinase C must therefore be
complemented with alternative experimental approaches that can distinguish
between the roles of PKCs and non-PKC DAG/phorbol-ester receptors in DAG
signalling processes. Detailed genetic analyses of
C1-domain-containing non-PKC DAG/phorbol-ester receptors in mammals
have proven to be the most promising approach in this respect. Depending on
the cellular system under investigation, RNAi is likely to be of comparable
potential.

Acknowledgements

We would like to thank the members of our laboratories for discussions and
invaluable contributions, in particular J.-S. Rhee and A. Betz for providing
the data shown in Figs
2 and
3. Work in the authors'
laboratories was generously supported by the Max-Planck-Society and the German
Research Foundation.

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